Synthesis and characterization of norbelladine, a precursor of Amaryllidaceae alkaloid, as an anti-inflammatory/anti-COX compound

Article abstract of DOI:10.1016/j.bmcl.2014.10.051

Rising ROS and systemic inflammation is often a serious concern in many disease conditions including obesity. Therefore, compounds with both anti-oxidant and anti-inflammatory activities are considered beneficial in preventing/treating several human chronic diseases. Norbelladine is an amine compound, a precursor for Amaryllidaceae alkaloids (e.g., belladine, crinamine, lycorine, and galanthamine) found in plants traditionally used for treating a variety of human diseases. However, little information is available about its potential health effects. Therefore, the amine was first synthesized, and its anti-oxidant and anti-inflammatory activities were investigated in this study. Also, the potential effects of the amine on NF-κB activation were investigated due to the critical involvement of ROS in the activation. Norbelladine was synthesized with more than 60% yield, analyzed by a HPLC method, and verified using NMR spectroscopic method. Then, its radical scavenging activity was investigated using DPPH- and superoxide radical assays. At the concentration of 10 μM, norbelladine was a compound able to quench DPPH-radical by 31% (P < 0.05) and reduce superoxide radicals from xanthine oxidase by 33% (P < 0.05). At the concentration of 0.25 μM, the amine also inhibited both COX-1 and COX-2 enzymes by 51% and 25% (P < 0.05), respectively. Furthermore, at the concentration of 10 μM, norbelladine inhibited NF-κB activation by 23% (P < 0.05). In summary, the data suggests that norbelladine may be a compound to quench radicals, inhibit COX enzymes as well as suppress NF-κB activation at relatively lower concentrations.

Full text of DOI:10.1016/j.bmcl.2014.10.051

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5381–5384
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and characterization of norbelladine, a precursor of
Amaryllidaceae alkaloid, as an anti-inflammatory/anti-COX
compound
⇑
Jae B. Park
Diet, Genomics, and Immunology Laboratory, Bldg. 307C, Rm. 131, BHNRC, ARS, USDA, Beltsville, MD 20705, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Rising ROS and systemic inflammation is often a serious concern in many disease conditions including
obesity. Therefore, compounds with both anti-oxidant and anti-inflammatory activities are considered
beneficial in preventing/treating several human chronic diseases. Norbelladine is an amine compound,
a precursor for Amaryllidaceae alkaloids (e.g., belladine, crinamine, lycorine, and galanthamine) found
in plants traditionally used for treating a variety of human diseases. However, little information is avail-
able about its potential health effects. Therefore, the amine was first synthesized, and its anti-oxidant and
anti-inflammatory activities were investigated in this study. Also, the potential effects of the amine on
NF-jB activation were investigated due to the critical involvement of ROS in the activation. Norbelladine
was synthesized with more than 60% yield, analyzed by a HPLC method, and verified using NMR spectro-
scopic method. Then, its radical scavenging activity was investigated using DPPH- and superoxide radical
Received 22 July 2014
Revised 10 October 2014
Accepted 17 October 2014
Available online 22 October 2014
Keywords:
Norbelladine
Chemical synthesis
NMR
Radicals
COX
NF-jB
assays. At the concentration of 10
(P < 0.05) and reduce superoxide radicals from xanthine oxidase by 33% (P < 0.05). At the concentration of
.25 M, the amine also inhibited both COX-1 and COX-2 enzymes by 51% and 25% (P < 0.05), respec-
tively. Furthermore, at the concentration of 10 M, norbelladine inhibited NF- B activation by 23%
P < 0.05). In summary, the data suggests that norbelladine may be a compound to quench radicals, inhi-
lM, norbelladine was a compound able to quench DPPH-radical by 31%
0
l
l
j
(
bit COX enzymes as well as suppress NF-
j
B activation at relatively lower concentrations.
Published by Elsevier Ltd.
Obesity is a serious health condition often associated with sev-
proteins including cyclooxygenase-2 (COX-2). Especially related
to COX-2 expression, several data even suggest that the ROS-mod-
ified molecules may act as endogenous inducers of COX-2 respon-
sible for increasing prostaglandin production during inflammatory
eral chronic diseases such as diabetes, cardiovascular disease,
hypertension and chronic kidney diseases.1 Particularly, visceral
obesity is strongly associated with increased reactive oxygen spe-
cies (ROS) and subclinical systemic inflammation, which are con-
sidered as major causes for developing several obesity-induced
,2
9
–12
and immune responses.
Cyclooxygenases (prostaglandin H
synthase or PGHS) are enzymes with both cyclooxygenase and
3
,4
13–15
chronic diseases. In fact, ROS are normal by-products of mito-
chondrial respiratory chain activity.5 However, in association
with inadequate antioxidant capacity and some disease conditions,
the elevated levels of ROS can cause significant consequence to cel-
lular signals and components such as lipids, proteins, and DNA,
thereby shifting cell homeostasis.7 For instance, ROS activate
nuclear factor kB (NF-kB) via IkB kinase (IKK) through phosphoin-
ositide 3-kinase (PI3K) and other processes.9 NF-kB with other
proteins eventually leads to activating several transcription-associ-
ated factors such as AP-1 and CREB-binding protein, presumably
promoting the expression of multiple inflammation-related
peroxidase activities,
and there are two major forms of cyclo-
,6
oxygenases (COX-1 and COX-2). COX-1 is constitutively expressed
in numerous cell types, meanwhile the expression of COX-2 is tran-
siently induced by a variety of stimuli such as phorbol esters, lipo-
1
6,17
polysaccharides, and cytokines.
The COX enzymes catalyze the
,8
conversion of arachidonic acid to prostaglandin H2, the precursor
of important biological prostanoid mediators such as prostaglan-
dins, prostacyclin and thromboxane. By producing a variety of
prostaglandins, the enzymes are deeply involved in pathophysio-
,10
1
7
logical processes such as inflammation and pain. Since ROS and
COX have been considered to play significant roles in the initiation
and/or progression of several chronic human diseases, there has
been considerable research effort in order to find effective
compounds with both anti-ROS and anti-COX activities.
⇑
Tel.: +1 301 504 8365; fax: +1 301 504 9456.
E-mail address: jae.park@ars.usda.gov
http://dx.doi.org/10.1016/j.bmcl.2014.10.051
960-894X/Published by Elsevier Ltd.
0

5
382
J. B. Park / Bioorg. Med. Chem. Lett. 24 (2014) 5381–5384
Figure 1. Chemical structures of norbelladine.
Norbelladine is a precursor for Amaryllidaceae alkaloids (e.g.,
belladine, crinamine, lycorine, and galanthamine) found in several
plants such as Galanthus nivalis, Leucojum aestivum, Narcissus tazet-
ta, Nerine bowdenii, and Pancratium maritimum.1
8,19
Although the
potential effects of the Amaryllidaceae alkaloids have been studied
2
0,21
in a great number,
the potential health effects of norbelladine
Figure 3. DPPH-radical scavenging activity of norbelladine. DPPH assay was
performed at the concentrations of norbelladine, vanillic acid and p-hydroxyben-
has not been investigated in detail. Therefore, in this study, nor-
belladine (Fig. 1) was prepared via the chemical synthesis method
described in Supporting information section. The synthesis was
relatively simple, and the yield was greater than 60%. The synthe-
sized product was purified by HPLC, and analyzed using NMR spec-
troscopic methods (NMR data were provided in Supporting
information section). A HPLC method was developed using the
purified norbelladine as a standard. The amine was determined
using the high-performance liquid chromatography (HPLC)
equipped with Nova-Pak C18 column and an electrochemical detec-
tor. Since an electrochemical detector is used as a principal detec-
tor in this HPLC method, the electro-activities of the compound
were measured using the detector. Several different buffer condi-
tions were first tested to improve the resolution of the amine,
and a gradient condition was selected for optimizing separation,
detection and preparation of samples as described in Supporting
information section. The chromatograms of the amine standard
zoic acid (0, 5, 10, 20
calculated using SigmaPlot 11.0 (Holm-Sidak method; (P < 0.05)). The mark ( )
l
M). Data are presented as mean ± SD (n = 8). P value was
⁄
indicates a significant change compared to control samples.
The radical scavenging activity of norbelladine was determined
using a stable radical DPPH assay. The DPPH radical is reduced to a
colorless product as norbelladine scavenges the radical, and the
reduction can be measured by a UV–visible plate reader. As shown
in Figure 3, norbelladine showed relatively strong DHHP-radical
scavenging activity. Interestingly, benzoic moiety with hydroxyl
groups at the 3- and 4-positions showed better DHHP-radical scav-
enging activity than phenolics with one or modified hydroxyl
group such as p-hydroxybenzoic acid and vanillic acid (Fig. 3). This
suggests that intact hydroxyl groups at the 3- and 4-positions of
benzoic moiety in norbelladine may be critical in scavenging
DPPH-radical.
(
100 pmol) are shown with two precursors (tyramine and proto-
Since norbelladine was able to scavenge DHHP-radicals, its abil-
ity to quench superoxide radical was further investigated using
catechuic aldehyde) in Figure 2.
Figure 2. HPLC chromatograms of tyramine (A), protocatechuic aldehyde (B) and norbelladine (C). All three chemicals (100
liquid chromatography (HPLC), and detected by an electrochemical detector. The mark ( ) indicates an injection peak.
qmol) was injected into a high-performance
⁄

J. B. Park / Bioorg. Med. Chem. Lett. 24 (2014) 5381–5384
5383
Figure 4. Effects of norbelladine on ROS from xanthine/xanthine-oxidase. ROS
production was inhibited in a cell-free xanthine/xanthine-oxidase system using
different concentrations of norbelladine (0, 5, 10, 20 M). Data are presented as
l
mean ± SD (n = 8).
P
value was calculated using SigmaPlot 11.0 (Holm-Sidak
⁄
method; (P < 0.05)). The mark ( ) indicates a significant change compared to
Figure 6. Effects of norbelladine on COX-2 enzyme. Control (1), NS-398 (2), and
norbelladine (3). The amides or NS-398 were added to the samples, and the reaction
mixtures were incubated at room temperature (in the dark) for 10 min. Following
the incubation, COX-2 activity was measured according to the kit’s protocol using a
luminometer. The mark ( ) indicates a significant reduction compared to control
samples.
control samples.
xanthine/xanthine oxidase system. Xanthine oxidase (XO) cata-
lyzes hypoxanthine to xanthine and further catalyzes xanthine to
⁄
2
2
uric acid. During the catalysis, XO generates superoxide, a pow-
erful reactive oxygen species, leading to cell degeneration and
death. XO is found predominantly in the liver and intestine, but
levels of circulating XO can increase dramatically with some dis-
ease conditions including diabetes and hypertension.22 As shown
in Figure 4, norbelladine was able to scavenge superoxide radical
from xanthine/xanthine oxidase system by 33% (P < 0.05) at a con-
(
e.g., aspirin) are known to inhibit inflammation and platelet acti-
vation via preventing the conversion of arachidonic acid (AA) to
13–15
thromboxane A2 (TXA2) and others.
belladine was a compound able to inhibit COX-1 enzyme by 51%
P < 0.05) at the concentration of 0.25 M. On comparison with a
As shown in Figure 5, nor-
(
l
well-known COX-1 inhibitor (aspirin), norbelladine was able to
inhibit COX-1 to a greater extent than the inhibitor. Although
COX-1 enzyme is primarily involved in platelets, the effect of nor-
belladine on COX-2 was also investigated, because COX-2 is likely
to be involved in many important physiological processes related
to inflammation.
centration of 10 lM comparable to the control, confirming that the
amine compound may be a good superoxide-radical scavenger.
These data clearly indicated that norbelladine is able to scavenge
superoxide-radicals generated from XO.
Potential effect of norbelladine on COX-1 was investigated as an
inhibitory mechanism of inflammation, because COX-1 inhibitors
14,15
Like the COX-1, norbelladine was also a com-
pound to inhibit COX-2 enzyme by 25% (P < 0.05) at the concentra-
tion of 0.25 M. Compared to NS-398 (a COX-2 specific inhibitor),
l
norbelladine was able to inhibit COX-2 as much as NS-398 (Fig. 6).
9
Figure 5. Effects of norbelladine on COX-1 enzyme. Control (1), Aspirin (2), and
norbelladine (3). The amides or aspirin were added to the samples, and the reaction
mixtures were incubated at room temperature (in the dark) for 10 minutes.
Following the incubation, COX-1 activity was measured according to the kit’s
Figure 7. Effects of norbelladine on NF-kB activation. THP-1 cells (2 Â 10 ) were
incubated for 15 min with several concentrations of norbelladine (0, 1, 10, 20 lM),
then treated with LPS (0.5
l
g/ml) for 16 h. The cells were harvested and washed
with PBS twice. The NF-
j
B assay was performed using Cayman’s NF-
j
B (p65)
⁄
⁄
protocol using a luminometer. The mark ( ) indicates a significant reduction
transcription factor assay kit (Ann Arbor, Michigan). The mark ( ) indicates a
compared to control samples.
significant reduction compared to control samples.

5
384
J. B. Park / Bioorg. Med. Chem. Lett. 24 (2014) 5381–5384
These data suggest that norbelladine is a compound able to inhibit
both COX-1 and 2 enzymes.
found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2014.10.051.
As mentioned previously, ROS is deeply involved in activating
NF-
jB and the activation is closely associated with expressing
References and notes
numerous inflammation-related proteins including cyclooxygen-
9
,10
1. Kaur, J. Cardiol. Res. Pract. 2014, 2014, 1.
ase-2 (COX-2).
NF- B activation were investigated. For the experiment, THP-1
cells (2 Â 10 ) were treated with several concentrations of norbell-
adine (0, 1, 10, 20 M), then treated with LPS as described in Sup-
porting information section. As shown in Figure 7, norbelladine
inhibited the NF- B activation induced by LPS by 23% (P < 0.05)
at the concentration of 10 M. The data suggest that the amine
may inhibit COX I and II enzymes as well as suppress COX II
expression via inhibiting NF- B activation. This study suggests that
Therefore, the potential effects of the amine on
2
3
.
.
Hajer, G. R.; van Haeften, T. W.; Visseren, F. L. Eur. Heart J. 2008, 29, 2959.
Schulz, E.; Gori, T.; Münzel, T. Hypertens. Res. 2011, 34, 665.
j
9
4. Dali-Youcef, N.; Mecili, M.; Ricci, R.; Andrès, E. Ann. Med. 2013, 45, 242.
5
6
7
8
.
.
.
.
Wei, Y. H.; Lu, C. Y.; Lee, H. C.; Pang, C. Y.; Ma, Y. S. Ann. N.Y. Acad. Sci. 1998, 854,
55.
Richter, C.; Gogvadze, V.; Laffranchi, R.; Schlapbach, R.; Schweizer, M.; Suter,
M.; Walter, P.; Yaffee, M. Biochim. Biophys. Acta 1995, 1271, 67.
l
1
j
Cutler, R. G.; Plummer, J.; Chowdhury, K.; Heward, C. Ann. N.Y. Acad. Sci. 2005,
l
1
055, 136.
Mena, S.; Ortega, A.; Estrela, J. M. Mutat. Res. 2009, 674, 36.
j
9. Uchida, K. Mol. Cells 2008, 25, 347.
10. Kanayama, M.; Yamaguchi, S.; Shibata, T.; Shibata, N.; Kobayashi, M.; Nagai, R.;
Arai, H.; Takahashi, K.; Uchida, K. J. Biol. Chem. 2007, 282, 24166.
norbelladine may be a compound able to provide significant anti-
oxidant and anti-inflammatory effects via scavenging radicals
1
1. Upmacis, R. K.; Deeb, R. S.; Hajjar, D. P. Prostaglandins Other Lipid Mediat. 2006,
0, 1.
12. Zou, M. H. Prostaglandins Other Lipid Mediat. 2007, 82, 119.
and inhibiting COX enzymes, furthermore inhibiting NF-
tion at relatively lower concentrations.
jB activa-
8
1
1
1
1
1
3. Smith, W. L.; Meade, E. A.; DeWitt, D. L. Ann. N.Y. Acad. Sci. 1994, 18, 136.
4. Lefkowith, J. B. Am. J. Med. 1999, 31, 43S.
5. Simon, L. S. Curr. Opin. Rheumatol. 1997, 9, 178.
6. Kim, S. F.; Huri, D. A.; Snyder, S. H. Science 2005, 23, 1966.
7. Sharma, S.; Zhu, L.; Yang, S. C.; Zhang, L.; Lin, J.; Hillinger, S.; Gardner, B.;
Reckamp, K.; Strieter, R. M.; Huang, M.; Batra, R. K.; Dubinett, S. M. J. Immunol.
Acknowledgment
This study was funded by USDA (Project number 1235-51000-
54-00D).
0
2005, 15, 813.
Disclosures: There is nothing to disclose about financial, consult-
ing, and personal matters related to this Letter.
18. Mann, J. D.; Fales, H. M.; Mudd, S. H. J. Biol. Chem. 1963, 238, 3820.
1
2
9. El Tahchy, A.; Ptak, A.; Boisbrun, M.; Barre, E.; Guillou, C.; Dupire, F.; Chrétien,
F.; Henry, M.; Chapleur, Y.; Laurain-Mattar, D. J. Nat. Prod. 2011, 74, 2356.
0. Marco, L.; Do Carmo Carreiras, M. Recent Pat. CNS Drug Discov. 2006, 1, 105.
Supplementary data
21. Jin, Z. Nat. Prod. Rep. 2013, 30, 849.
2. Pacher, P.; Nivorozhkin, A.; Szabó, C. Pharmacol. Rev. 2006, 58, 87.
2
Supplementary data (experimental details and compound char-
acterization for norbelladine) associated with this article can be